Cytogenetic studies in three octopods, Octopus minor, Amphioctopus fangsiao, and Cistopus chinensis from the coast of China

CompCytogen |2(3): 373-386 (2018) COMPARATIVE *reerrerevedorenacessiours

doi: 10.3897/CompCytogen.v | 2i3.25462 A Cyto genetics

http://compcytogen.pensoft.net International journal of Plant & Animal Cytogenetics,
i

Karyosystematics, and Molecular Systematics

Cytogenetic studies in three octopods, Octopus minor,
Amphioctopus fangsiao, and Cistopus chinensis
from the coast of China

Jin-hai Wang'’, Xiao-dong Zheng!”

| Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao 266003, China 2. Key
Laboratory of Mariculture, Ocean University of China, Ministry of Education, China

Corresponding author: Xiao-dong Zheng (xdzheng@ouc.edu.cn)

Academic editor: V. Specchia | Received 3 April 2018 | Accepted 12 August 2018 | Published 4 September 2018
http://zo0bank.ore/4B697 LF2-34FC-4A20-AA0 1-OD0EB22B75A8

Citation: Wang J-h, Zheng X-d (2018) Cytogenetic studies in three octopods, Octopus minor, Amphioctopus fangsiao,
and. Cistopus chinensis from the coast of China. Comparative Cytogenetics 12(3): 373-386. https://doi.org/10.3897/
CompCytogen.v12i3.25462

Abstract

To provide markers to identify chromosomes in the genome of octopods, chromosomes of three octopus
species were subjected to NOR/C-banding. In addition, we examined their genome size (C value) to sub-
mit it to the Animal Genome Size Database. Silver staining revealed that the number of Ag-nucleoli was 2
(Octopus minor (Sasaki, 1920)), 2 (Amphioctopus fangsiao (VP Orbigny, 1839)) and 1 (Cistopus chinensis
Zheng et al., 2012), respectively, and the number of Ag-nucleoli visible was the same as that of Ag-NORs
on metaphase plates in the same species. In all analyzed metaphases, Ag- NORs were mainly located termi-
nally on the long arms of chromosomes 3 (3") of O. minor and on the short arms of chromosomes 4 (4")
of A. fangsiao, whereas only one of the chromosomes 23 (23) was found Ag-NORs of C. chinensis. C-
bands were localized predominantly in the centromeric regions of chromosomes in the three species, while
other conspicuous stable C-bands were observed in terminal regions, including the Ag-NORs. That means
these three chromosome pairs (3", 4° and 23") could be considered species-specific cytogenetic markers.
The mean C'values of O. minor, A. fangsiao and C. chinensis were 7.8140.39 pg (0.070 pg per unit length),
8.3140.18 pg (0.068 pg per unit length) and 5.29+0.10 pg (0.038 pg per unit length), respectively, and
results showed that C’ values of the three species were not proportional to the relative length of the chro-
mosomes. These cytogenetic characteristics will provide more theoretical foundation for further researches

on chromosome evolution in octopods.
Keywords

octopods, karyotype, Ag-NORs, C-bands, genome size, flow cytometry

Copyright Jin-hai Wang, Xiao-dong Zheng. This is an open access article distributed under the terms of the Creative Commons Attribution License
(CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

374 Jin-hai Wang @ Xiao-dong Zheng / Comparative Cytogenetics 12(3): 373-386 (2018)

Introduction

Genetics and cytology combine to establish cytogenetics, mainly from the perspective of
cytology, especially from a chromosome structure and function as well as the relationship
between chromosomes and other organelles, to elucidate the mechanism of inheritance
and variation. Cytogenetic analysis usually includes chromosome karyotype, band type,
flow karyotype analysis and fluorescence in situ hybridization. Previous published reviews
on chromosomal studies of molluscs were greatly increased since cytogenetic techniques
including silver-staining, C-and G-banding and have begun to be applied to molluscan
chromosomes (Thiriot-Quiévreux 2003). Although these techniques have been widely
used in the study of shellfish chromosomes, there are no reports on the cephalopods.

Octopods, such as Octopus minor (Sasaki, 1920), Amphioctopus fangsiao (d’Orbigny,
1839) and Cistopus chinensis Zheng et al., 2012 are cephalopod species. ‘The previous
chromosome analysis of cephalopods had revealed 2n=G60 or 92 in most species studied,
including O. minor, A. fangsiao, C. chinensis, two sepiids (Sepia esculenta and S. lycidas
Gray, 1849) and three loliginids (Heterololigo bleekeri Natsukari, 1984, Sepioteuthis les-
soniana Bainville, 1824 and Photololigo edulis (Hoyle, 1885)) (Gao and Natsukari 1990,
Adachi et al. 2014, Wang and Zheng 2017), although Nautilus macromphalus Sowerby,
1849 had 2n=52 chromosomes (Bonnaud et al. 2004), and the studies led by Papan and
Jazayeri reported the chromosome number of S. arabica Massy, 1916 and S. pharaonis
Ehrenberg, 1831 was 2n=48 (Papan et al. 2010, Jazayeri et al. 2011). However, there
are only a few studies on the cephalopod chromosomes in reported publications, and
there are no researches on the band type in these species. Adachi et al. (2014) first tried
to use fluorescence in situ hybridization on the cephalopod chromosomes and suggested
that the telomere sequence of O. areolatus de Haan, 1839-1841 was (TTAGGG)n, but
there was a lack of complete and clear metaphases in the report. Due to the restriction
of the embryo acquisition, and the number of cephalopod chromosomes up to 60, it is
difficult to obtain an ideal metaphase. All these factors seriously restrict the cytological
study of cephalopods. In a previous published paper, we gave a detailed overview of the
existing cephalopod chromosome information, including the genetic relationship anal-
ysis based on evolutionary distance (Wang and Zheng 2017). The present study used
gills as materials, and through a large number of repeated tests, the ideal metaphases
with NORs and C-bands were obtained based on the previous study.

As an important part of the study of cytogenetics, more and more genome sizes
(C values) have been revealed. Although the genome sizes of 281 mollusks have been
submitted to the Animal Genome Size Database (http://www.genomesize.com. Ac-
cessed December 25, 2017) while there just have been 6 species of cephalopod C values
that can be obtained from the database: O. bimaculatus (Hinegardner 1974), O. bi-
maculoides (Albertin et al. 2015), O. vulgaris (Packard and Albergoni 1970), Euprymna
scolopes (Adachi et al. 2014), Loligo plei (Hinegardner 1974) and Loliginidae sp. (Mir-
sky and Ris 1951). These C’ values were estimated based on bulk fluorometric assay and
feulgen image analysis densitometry. As genomic and transcriptomic sequencing is car-
ried out in cephalopods, more and more cephalopod genome sizes have been revealed

Cytogenetic studies in three octopods 375

Table |. The published information of cephalopod genome size. BA: Biochemical Analysis, FCM: Flow
Cytometry, BFA: Bulk Fluorometric Assay, CGS: Complete Genome Sequencing, FIA: Feulgen Image

Analysis Densitometry.

Species Origin ae es Method. Ene References
Packard and
O. vulgaris sperm Not specified | BA/CGS | 5.15 pg/2.5-5 Gb | Albergoni 1970;
Albertin et al. 2012
O. minor Haemocytes G. domesticus FCM 7 .82+0.56 pg This study
O. bimaculatus sperm SOONER CEP Orn BFA 4.30 pg Hinegardner 1974
purpuratus
’ ; ; Albertin et al. 2012,
O. bimaculoides Not specified | Not specified | BFA/CGS| 2.93 pg/3.2 Gb 2015
A, fangsiao Haemocytes G. domesticus FCM 8.2340.42 pg This study
C. chinensis Haemocytes G. domesticus FCM 5.13+0.38 pg This study
H.. maculosa ~ ~ CGS 4.5 Gb Albertin et al. 2012
S. officinalis - ~ CGS 4.5 Gb Albertin et al. 2012
L. plei sperm S. purpuratus BFA 2.80 pg Hinegardner 1974
L. pealeii — - CGS 2.7 Gb Albertin et al. 2012
E. scolopes gee G. domesticus. | FIA/CGS | 3.75 pg/3.7 Gb nie ieee ee )

Sperm

Yoshida et al. 2011
Albertin et al. 2012
Yoshida et al. 2011

2.1 Gb
4.5 Gb
2.84.2 Gb

I. paradoxus
A. dux
N. pompilius

— CGS
— CGS

by complete genome sequencing such as NV. pompilius, Architeuthis dux, Hapalochlaena
maculosa, E. scolopes, Idiosepius paradoxus, L. pealeii, S. officinalis, etc (Yoshida et al.
2011, Albertin et al. 2012) (Table 1). Besides, Adachi et al. (2014) examined the C
values of O. ocellatus and O. vulgaris based on flow cytometry. Although there are
many methods to detect C’values, we choose flow cytometry for the convenience, rapid
analysis and relative accuracy of the sample preparation (Gokhman et al. 2017).

To develop octopus chromosome markers, the present research has first completed
NOR/C-banding. Also we examined their C values to submit to the Animal Genome
Size Database. ‘This is a basic work for molecular cytogenetic research of octopods. It
is expected to lay a solid theoretical foundation for further researches on chromosome
evolution in octopods.

Material and methods
Ag-staining of the NORs and C-bands
Collection of samples and chromosome slides preparation based on the previous pub-

lished paper (Wang and Zheng 2017). The nucleoli in interphase and the NORs in

metaphase were visualized using rapid silver nitrate staining using the previous meth-
p g rap g g p

376 Jin-hai Wang @ Xiao-dong Zheng / Comparative Cytogenetics 12(3): 373-386 (2018)

ods (Howell and Black 1980). C banding were carried out following the protocols of
Sumner (1972) with some modifications. The dyed chromosome slides were detected
under a light microscope with an oil lens (Leica MC170 HD, Germany).

Genome size (C value)

Preparation of cell suspension

Ten individuals (5 males and 5 females) of each species were used for collecting hemo-
lymph. All subjects were handled according to the guidelines put forth by the EU
Directive 2010/63/EU for cephalopod welfare (Fiorito et al. 2014). Before dissecting,
all octopods should be anesthetized using 7.5% magnesium chloride (MgCl,) solution
(Messenger et al. 1985) until unconscious.

Then hemolymph was withdrawn from the heart or cephalic aorta of each octopus
using a disposable syringe, and the hemolymph was immediately transferred into a
1.5 ml centrifuge tube containing precooled (4 °C) phosphate-buffered saline (PBS)
(v/v=1:1). Mixed samples were centrifuged for 12 min at 300g and 4 °C, and then
the hemocytes were resuspended twice in PBS according to above. ‘The last suspen-
sion containing 300 pl PBS was added to another tube containing 900 pl precooled
(—20 °C) anhydrous ethanol (v/v=1:3), fixing at least 3 hours at 4 °C. The fixed cell
was washed twice in PBS after it was suspended with PBS up to 1 ml. Before the flow
cytometry detection, a moderate propidium iodide solution (PI, 20-30 pg/ml) was
added to the suspensions, staining for 2 hours at 4 °C in dark.

Flow cytometry analysis

Previous studies have shown that the genome size (C value) of chickens (Gallus do-
mesticus) was 1.25 pg (Tiersch et al. 1989, Adachi and Okumura 2012, Adachi et al.
2014). Here we determined C’' values of octopods using this chicken red blood cells
as internal standard, and the standard sample was purchased from BD company
(DNA QC Particles).

C value was measured using a model PA flow cytometer (Beckman Coulter
Cytomics FC 500 MPL), in principle, at least 15,000 cells were measured in each
sample. The blue light of 488 nm was first excited, and the fluorescence of PI
was detected by the emission wave length of 625410 nm. The present study used
chicken standard sample as calibration instrument, and then used it as the internal
standard, by comparing multiple relationships between the standard sample (chick-
en red blood cells) and the pending sample (octopus hemolymph) peak, calculating
the C values.

Cytogenetic studies in three octopods Ove

Figure |. Ag-nucleoli of interphase. a 1, 2 or 3 normal nucleolus organizer regions (NORs) in interphase

of O. minor and A. fangsiao, and the two species mainly contain 2 NORs b 1 or 2 NORs in interphase of
C. chinensis, and most of them contain 1 NORs . Scale bar: 5 um.

Results
Banding analysis

After silver staining was performed, the number of Ag-nucleoli was between 1-3 in in-
terphase nuclei of three species (Fig. 1). We randomly selected 200 interphase nuclei to
calculate the number of nucleolar organizer regions for each species. Among the scored
interphases 24% had 1 nucleolus, and 61% had 2 nucleoli, and 10% had 3 nucleoli
and 5% had more than 3 nucleoli in O. minor species. Twenty-four metaphases were
counted indicating there were 2 Ag-NORs, then 7 of them were selected for karyotype
analysis and Ag-NOR loci were located on the long arms of metacentric chromosomes
3 (3"). In A. fangsiao species, 38 of 200 interphase nuclei showed 1 Ag-nucleolus, then
146 of them contained 2 nucleoli and 16 of them had 3 to 5 nucleoli. Eighteen meta-
phases were counted and 7 of them were selected for karyotype analysis, showing there
were 2 Ag-NORs and Ag-NOR loci were located on the short arms of the metacentric
chromosomes 4 (4°). For C. chinensis species, there were mainly 1 nucleolus (up to
68%), followed by 2 nucleoli accounting for about 21%, while a small proportion had
3-5 nucleoli. Selected 13 scored metaphases indicated that there were only 1 Ag- NOR
and then 7 karyotypes were analyzed suggesting that Ag-NOR loci were located on
long arms of the subtelomeric chromosome 23 (23") (Fig. 2).

The C-band karyotype analysis indicated that there were 31 C-bands (O. minor),
25 C-bands (A. fangsiao) and 25 C-bands (C. chinensis) in three species of octopods,
respectively. C-bands were consistently localized in the centromeric regions of most
chromosomes in the three species, but which varied in size (Fig. 3d—f), and in C. chin-
ensis, the C-bands of long arms were smaller those of O. minor and A. fangsiao. In par-
ticular, several other steady C-bands were located on terminal region of chromosomes

378 Jin-hai Wang @ Xiao-dong Zheng / Comparative Cytogenetics 12(3): 373-386 (2018)

AE XR KX AK HK KA AX HH XK XX KA

RA AX KK AR NK WERK KKK MS MA KA

13 «14 15 16 #17 #18 19 #20 21 22 23 24
AN AR AA me mm ne —
25 «26 27) 06-280 29) 30

a

XA YC RE WD AS KK AK RK ede xx Das ae
1 ? 3 4 5 6 T 8 9 10 11 12
mx xx xe oh HR BAAR KK HM I oe

18 19 20 ?1 22 23 24
ARG AA IR Os ma a ba

25 26 27 28 29 30 d —
MD AK AK Dy EE WRK AB WH KK poe mR
1 2 j «« § & 8 9 0 1M 12
Pi

paw wwe Dh wt ws

M AG ad as A ar “Tt f

23 ® 24 25 26 27 28 «29 30 iF

Figure 2. Karyotypes and NOR-bearing chromosomes from three species of octopod gills. a The metaphase
plate of O. minor b Karyogram of O. minor from (a) showed that Ag-NOR loci were located on the long
arms of metacentric chromosome 3 € The metaphase plate of A. fangsiao d Karyogram of A. fangsiao from (€)
showed that Ag-NOR loci were located on the short arms of metacentric chromosome 4 e The metaphase
plate of C. chinensis f Karyogram of C. chinensis from (e) showed that Ag-NOR loci were located on long

arms of subtelomeric chromosome 23. Arrows indicate the NOR-bearing chromosomes. Scale bars: 5 um.

3 (3") for O. minor, chromosomes 4 (4) for A. fangsiao and chromosomes 23 (23"')
for C. chinensis, which was coincident with positive Ag-NOR loci, except for chromo-
somes 23 in C. chinensis, where only one of the chromosomes 23 was found to have an
Ag-NOR (Fig. 2f). Besides, various C-bands were observed on the long arms of chro-
mosome pairs 2 (proximal terminal region), 7 (interstitial region) and 25 (proximal
terminal region) in O. minor, chromosome pairs 3 (interstitial region) and 17 (inter-
stitial region) in A. fangsiao and chromosome pair 4 (terminal region) in C. chinensis.

Combined with the results of silver staining and C-banding, although C-bands
were localized predominantly in the centromeric regions of chromosomes in the three
octopus species, other conspicuous C-bands were observed in terminal regions, includ-
ing the Ag-NORs. Therefore, we can select chromosome pairs 3, 4 and 23 as effective
species-specific markers to distinguish the three octopods.

C value

In data analysis, we selected about 15,000 cells per sample, then fluorescence in-
tensity representing the relative DNA content was recorded. Figure 4a showed the

Cytogenetic studies in three octopods mH)

QO. minor

a a ee) ee |
a
. M | —-&® ax ae we ee ue nx
"4 “>
” P + ax ee et re pe hd
oS Mee
+g re SM | is Aa he
ae =
“Ap tir* ST | Anan
‘vs T|~—™~ aa " ve a
A. fangsiao
kV ye", * y | Me ME Me RN RR RH RR RS
a lg ape x ~~ «om OU
c o
att 1.¢ SM kx ue Se An AA
oar = e, , | Se i me
~ + = T| an an AR OM fk fe b
C. chinensis P a9 34> <¢ «> AB-MK 24 kD
~
aera nedhadnaietnennaetne
Gye? ons
* = 7
Pa Pe sM| AR M&O
“ 4s ST | Ad >{ Ax AA
4 = C
T | ae a+ # ‘

M

I | |
I I I
| I I
I | |
I | I
ZT TTT LILT rrr rr rrTrrIxsrTxrxrrrFIAFTxrIX Za
; : :
I | |
- oF

= M

ba RD es ee i aah ie? ll dei QU et> Se Ei
|_|

M

f

Figure 3. C-banding karyotypes arranged from mitotic metaphases of O. minor (a), A. fangsiao (b)

1 I
I I
| |
| I
XXYTrrrYTrTrXrI.s xX: XT xrrVrrrr:Es:
I |
| I
I I
| I
F a!

and C. chinensis (€) and diagrams of C-banding (d, e, f). Black dots representing the chromosomes with
C bands/heterochromatin blocks. Scale bars: 5 pm.

380 Jin-hai Wang @ Xiao-dong Zheng / Comparative Cytogenetics 12(3): 373-386 (2018)

X-Mean=10, 4

675

G. domesticus

O. minor

a
i 10° 10! 10° 10
FSLin Relative DNA content

10° 40" 107 40?
Relative DNA content

441
466

A. fangsiao

em orre}
a

10° 107 _— OO a TT
Relative DNA content 10° 10" 10° 40°
Relative DNA content

Figure 4. Flow cytometry profiles of relative fluorescence intensity of propidium iodide (PI) in octopus
hemocytes of b O. minor € A. fangsiao and d C. chinensis with G. domesticus a as standard (St). Scatter
plots display the quality and number of cell mass of standard samples and test samples, and the peak fig-
ures show the relative DNA content of each sample. b, c and d are just a representative graph of the three

species of octopus samples. CN, the number of cells; X-MEAN, mean fluorescence intensity.

number of cells (CN=14699) and mean fluorescence intensity (X-Mean=10.4) of the
internal standard and other three representative results of O. minor (CN=10385, X-
Mean=64.5, Fig. 4b), A. fangsiao (CN=14756, X-Mean=72.3, Fig. 4c) and C. chinensis
(CN=14655, X-Mean=43.8, Fig. 4d) also were enumerated to estimate the DNA con-
tent. The results showed the mean C’ values of O. minor, A. fangsiao and C. chinensis
were 7.81+0.39 pg (male 7.85+0.47 pg, female 7.76+0.32 pg), 8.3140.18 pg (male
8.33+0.25 pg, female 8.30+0.10 pg) and 5.29+0.10 pg (male 5.28+0.08 pg, female
5.29+0.12 pg), respectively. C. chinensis had the smallest C value, significantly lower
than O. minor (P < 0.05) and A. fangsiao (P < 0.05) (Table 2).

Based on our previous studies, the chromosome total relative lengths of O. minor,
A, fangsiao and C. chinensis were 112.33, 122.77 and 139.20. C. chinensis had the
largest relative length, followed by A. fangsiao and O. minor, which was not propor-
tional to the C'value. Obviously, C. chinensis had the smallest C value but the largest
chromosome relative length. The DNA content of the unit length chromosomes of
the three octopods was about 0.070 pg (O. minor), 0.068 pg (A. fangsiao) and 0.038
pg (C. chinensis) respectively. Results suggested that there was no significant positive
proportional relationship between the C value and the relative chromosome length.
Besides, this study analyzed the C’ values of 14 species of cephalopods, ranging of 2.20
to 8.23 pg (2.10—7.86 Gb), of which 1. paradoxus had the smallest genome size and

Cytogenetic studies in three octopods 381

Table 2. The results of C values from three species of octopods.

Species Sample no. C value (X+SE) /pg
G. domesticus 0 1125
2
male 3 70.4 8.46 7.85+0.47
4 Che 8.08
O. minor 5 65.5 7.87 7.8140.39
6
7 63.5 7.63
female 3 67.2 8.08 7.7640.32
9 66.1 7.94
male a oe 8.33+0.25
3 67.5 8.11
A, fangsiao : - 77 8.31+0.18
female S os: 8.30+0.10
i
8 69.0 8.29
1 43.8 5.26
male 2 44.6 5.36 5.28+0.08
3
C. chinensis 4 43.5 5.23 5.29+0.10
5 44.8 5.38
female - 45.0 541 5.29+0.12
7

the largest value from A. fangsiao. Overall, the average genome size of 6 species from
Octopoda (3.35—8.23 pg) was higher than that of 8 species from Sepiida and Teuthida
(2.20-4.71 pg).

Discussion

We first carried out silver staining (Ag-NOR) in octopus species, and the results
showed that C. chinensis had only one nucleolus organizer region (NOR) which was
located terminally at the long arms of a pair of homologous chromosomes. O. minor
and A. fangsiao had two NORs which located terminally on a pair of homologous
chromosomes. As an effective chromosome marker, polymorphisms in NORs can be
observed in interspecies or intraspecies comparisons, including the number, location
and size of sliver staining (Wang et al. 2015, Zalesna et al. 2017), even the geographi-
cal location and habitat differences can cause the diversity. However, many studies
have determined that the number of NORs in interphase is consistent with that on

382 Jin-hai Wang @ Xiao-dong Zheng / Comparative Cytogenetics 12(3): 373-386 (2018)

metaphase plates of the same species (Iizuka et al. 2013, Zalesna et al. 2017). Accord-
ing to the report of Okumura et al. (1999), NORs of the Haliotis discus hannai were
located at the end of two pairs of chromosome long arms, and it was also found in the
subcentral centromeric chromosome and the centromeric chromosome. Similarly, in
the later studies of abalone chromosomes from Wang et al. (2015) showed NOR sites
located on the 14 and 17 chromosomes, but at the end of the short arms of central
and submetacentric chromosomes also were found the sites. From the two studies, the
same species proved that the polymorphism of Ag-NOR bands was prevalent among
most species, including crustaceans, teleost fish, reptiles, mammals and other mol-
lusks (Babu and Verma 1985, Thiriot-Quiévreux and Insua 1992, Vitturi and Lafargue
1992, Cross et al. 2003, Britton-Davidian et al. 2011).

The number of C-bands in O. minor was larger than that of the other two octopus
species, which was consistent with the Ag-NORs. Although A. fangsiao and C. chin-
ensis had the same number of C-bands, while the former contained more interstitial
C-bands. Taking these two points into account, it is confirmed that the O. minor is
more advanced in evolution from the chromosome level. Almost all chromosomes of
three species of octopods can show C-bands in centromere regions, and it means that
heterochromatic blocks are evident in the pericentromeric regions of chromosomes,
which is consistent with the traditional view that the centromere region is mainly con-
sisted of heterochromatin. The C-banding results showed that the NOR regions of the
three species were all deep-stained C-bands, which also coincided with the common
assumption that the NOR regions were composed of heterochromatin. The stable C-
bands can be used as markers for chromosome identification, while the higher poly-
morphic C-band is not suitable as a marker for distinguishing chromosomes, but it can
be used as a genetic marker for the study of chromosome polymorphism. In present
study, chromosomal markers for identifying three species of octopods were developed
by Ag-NORs and C-bands, and it was effective means. Long before that, Martinez-
Lage et al. (1995) managed to separate the chromosomes of three shellfish by banding
techniques, which confirmed the reliability of this method.

In the present study, due to different survival pressure from geographical dis-
tribution (Zhang and Onozato 2004), the genome size of C. chinensis from South
is significantly less than that of O. minor and A. fangsiao from North. Adachi et al.
(2014) pointed out that the genome size of O. (A.) areolatus was 5.47 pg, then this
study showed the value was about 8.23 pg, significantly higher than the former. The
main reason may be the difference of samples or the existence of hidden species. Al-
though some studies had shown (Rakic et al. 2014) that the genome size was related
to ecological factors, that was not absolute. The diversity of genome size involved the
interaction of multiple factors and can not simply attribute the differences to the ex-
ternal environmental factors.

DNA is linear on the chromosome. According to Adachi et al. (2014), genome
size of O. (A.) areolatus and O. vulgaris was proportional to the relative chromosome
length, they determined the values of the two octopods was 5.47 pg and 3.50 pg,
respectively. The genome size of O. (A.) areolatus was about 1.5 times higher than

Cytogenetic studies in three octopods 383

that of O. vulgaris, and this ratio coincided with the ratio of chromosome length to
122.60/66.30. Even so, we can not simply consider the existence of ploidy between
the two, because the number of chromosomes is identical. Therefore, we speculate that
genome duplication may occur during the evolution of O. vulgaris, which leads to the
ploidy relationship. In contrast, current studies have found that the genome size of O.
minor, A. fangsiao and C. chinensis have no obvious linear relationship with their chro-
mosome length. Different methods to detect the genome size of the same species usu-
ally yield different results. In the reported cephalopod genomes, the genome size of O.
vulgaris, O. bimaculoides, and E. scolopes have been determined by biochemical analy-
sis, bulk fluorometric assay, complete genome sequencing and feulgen image analysis
densitometry (Table 1). Regardless of the cephalopod species, the obtained genome
size by complete genome sequencing is generally larger than other testing methods.
For example, using biochemical analysis method to get O. vulgaris genome size is 5.15
pg (Packard and Albergoni 1970), and the result of genome sequencing is 2.5 to 5.0
Gb, about 2.62 to 5.24 pg (Albertin et al. 2012). The O. bimaculoides genome size is
2.93 pg/3.2 Gb (about 3.35 pg) by bulk fluorometric assay (Albertin et al. 2012) and
genome sequencing (Albertin et al. 2015), respectively. Besides, the E. scolopes genome
size by feulgen image analysis (3.75 pg) (Gregory 2013) is less than that of genome
sequencing (3.7 Gb, about 3.87 pg) (Albertin et al. 2012). The main reason for these
results is that genome size obtained by genome sequencing contains a complete set
of nucleotide sequences, including non-coding sequences, and the increase of non-
coding sequences largely obscures the correlation between genome size and species
evolution complexity. In addition, the statistical analysis of cephalopod genome size is
mainly based on the existing basic data. More cephalopod genomes are needed to be
sequenced to further analyze the genomic characteristics of the population.

In conclusion, the present study combining a previously published paper (Wang
and Zheng 2017) highlights our increased knowledge of cephalopod cytogenetic stud-
ies. Up to now, cytogenetic studies of the cephalopods have stepped forward: Thirteen
species of cephalopod chromosome information have been reported, of which three are
related to silver staining and C-banding, also fourteen species of cephalopod genome
size or haploid DNA content have been revealed. What needs to be done next is the
location of the functional genes (such as sex related genes) on the chromosomes to
further deepen cytogenetic study of cephalopods.

Acknowledgments

We thank associate Prof. Mingzhuang Zhu, who helped us in the flow cytometer anal-
ysis. We thank Yaosen Qian, Qiaozhen Ke and Bing Cai for providing the O. minor
and C. chinensis individuals from Rongcheng and Ningde, China. We thank Dr. Diego
Orol Gémez for correcting the grammar of the article. This study was supported by
research grants from National Natural Science Foundation of China (No31672257)
and Key Development Plan of Shandong Province (2016GSF115014).

384 Jin-hai Wang @ Xiao-dong Zheng / Comparative Cytogenetics 12(3): 373-386 (2018)

References

Adachi K, Ohnishi K, Kuramochi T, Yoshinaga T, Okumura SI (2014) Molecular cytogenetic
study in Octopus (Amphioctopus) areolatus from Japan. Fisheries Science 80(3): 445-450.
https://doi.org/10.1007/s12562-014-0703-4

Adachi K, Okumura SI (2012) Determination of genome size of Haliotis discus hannai and
H.. diversicolor aquatilis (Haliotidae) and phylogenetic examination of this family. Fisheries
Science 78(4): 849-852. https://doi.org/10.1007/s12562-012-0518-0

Albertin CB, Bonnaud L, Brown CT, Crookes-Goodson WJ, da Fonseca RR, Di Cristo C,
Dilkes BP, Edsinger-Gonzales E, Freeman RM, Hanlon RT, Koenig KM, Lindgren AR,
Martindale MQ, Minx P, Moroz LL, Noed! MT, Nyholm SV, Ogura A, Pungor JR,
Rosenthal JJC, Schwarz EM, Shigeno S, Strugnell JM, Wollesen T, Zhang GJ, Ragsdale
CW (2012) Cephalopod genomics: a plan of strategies and organization. Standards in
Genomic Science 7(1): 175-188. http://dx.doi.org/10.4056/sigs.3 136559

Albertin CB, Simakov O, Mitros T, Wang ZY, Pungor JR, Edsinger-Gonzale E, Brenner S,
Ragsdale CW, Rokhsar DS (2015) The octopus genome and the evolution of cephalo-
pod neural and morphological novelties. Nature 524(7564): 220-224. https://doi.
org/10.1038/nature14668

Babu K, Verma R (1985) Structural and functional aspects of nucleolar organizer regions
(NORs) of human chromosomes. International Review of Cytology-a Survey of Cell Biol-
ogy 94: 151-176. https://doi.org/10.1016/S0074-7696(08)60396-4

Bonnaud L, Ozouf-Costaz C, Boucher-Rodoni R (2004) A molecular and karyological ap-
proach to the taxonomy of Nautilus. Comptes Rendus Biologies 327(2): 133-138. https://
doi.org/10.1016/j.crvi.2003.12.004

Britton-Davidian J, Cazaux B, Catalan J (2011) Chromosomal dynamics of nucleolar organ-
izer regions (NORs) in the house mouse: micro-evolutionary insights. Heredity 108(1):
68-74. https://doi.org/10.1038/hdy.2011.105

Cross I, Vega L, Rebordinos L (2003) Nucleolar organizing regions in Crassostrea angu-
lata: chromosomal location and polymorphism. Genetica 119(1): 65-74. https://doi.
org/10.1023/A:1024478407781

Fiorito G, Affuso A, Anderson DB, Basil J, Bonnaud L, Botta G, Cole A, D’Angelo L, De Gi-
rolamo P, Dennison N, Dickel L, Di Cosmo A, Di Cristo C, Gestal C, Fonseca R, Grasso F,
Kristiansen T, Kuba M, Maffucci E Manciocco A, Mark FC, Melillo D, Osorio D, Palumbo
A, Perkins K, Ponte G, Raspa M, Shashar N, Smith J, Smith D, Sykes A, Villanueva R, Tublitz
N, Zullo L, Andrews P (2014) Cephalopods in neuroscience: regulations, research and the
3Rs. Invertebrate Neuroscience 14(1):13—36. http://dx.doi.org/10.1007/s10158-013-0165-x

Gao YM, Natsukari Y (1990) Karyological studies on seven Cephalopods. Venus 49: 126-145.

Gokhman VE, Kuhn KL, Woolley JB, Hopper KR (2017) Variation in genome size and karyo-
type among closely related aphid parasitoids (Hymenoptera, Aphelinidae). Comparative
Cytogenetics 11(1): 97-117. https://doi.org/10.3897/CompCytogen.v1 1i1.10872

Gregory TR (2013) Animal Genome Size Database. http://www.genomesize.com

Hinegardner R (1974) Cellular DNA content of the Mollusca. Comparative Biochemistry and
Physiology Part A 47(2): 447-460. https://doi.org/10.1016/0300-9629(74)90008-5

Cytogenetic studies in three octopods 385

Howell WM, Black DA (1980) Controlled silver-staining of nucleolus organizer regions with
a protective colloidal developer: a 1-step method. Experientia 36(8): 1014-1015. https://
doi.org/10.1007/BF01953855

lizuka K, Matsuda Y, Yamada T, Nakazato T, Sessions SK (2013) Chromosomal localization
of the 18S and 28S ribosomal RNA genes using FISH and AgNO, banding in Hynobius
quelpaertensis, H. tsuensis and Onychodactylus koreanus (Urodela: Hynobiidae). Current
Herpetology 32(2): 89-101. https://doi.org/10.5358/hsj.32.89

Jazayeri A, Papan EK Motamedi H, Mahmoudi S (2011) Karyological investigation of Persian
Gulf cuttlefish (Sepia arabica) in the coasts of Khuzestan province. Life Science Journal
8(2): 849-852.

Martinez-Lage A, Gonzdlez-Tizén A, Méndez J (1995) Chromosomal markers in three spe-
cies of the genus Mytilus (Mollusca, Bivalvia). Heredity 74(4): 369-375. https://doi.
org/10.1038/hdy.1995.55

Messenger JB, Nixon M, Ryan KP (1985) Magnesium chloride as an anaesthetic for ceph-
alopods. Comparative Biochemistry and Physiology 82(1): 203-205. https://doi.
org/10.1016/0742-8413(85)90230-0

Mirsky AE, Ris H (1951) The desoxyribonucleic acid content of animal cells and its evolution-
ary significance. Journal of General Physiology 34(4): 451-462. https://doi.org/10.1085/
jgp.34.4.451

Okumura SI, Kinugawa S, Fujimaki A, Kawai W, Maehata H, Yoshioka K, Yoneada R,
Yamamori K (1999) Analysis of karyotype, chromosome banding, and nucleolus organizer
region of Pacific abalone, Haliotis discus hannai (Archaeo-gastropoda: Haliotidae). Journal
of Shellfish Research 18: 605-609.

Packard A, Albergoni V (1970) Relative growth, nucleic acid content and cell numbers of the
brain in Octopus vulgaris (Lamarck). Journal of Experimental Biology 52(3): 539-552.
Papan F, Jazayeri A, Ebrahimipour M (2010) The study of Persian Gulf cuttlefish (Sepia
pharaonis) chromosome via incubation of blood cells. Journal of American Science 6(2):

162-164.

Rakic T, Siljak-Yakovlev S, Sinzar-Sekulic J, Lazarevic M, Stevanovic B, Stevanovic V, Lakusic
D (2014) Morphological and genome size variations within populations of Edraianthus
graminifolius “Jugoslavicus” (Campanulaceae) from the central Balkan peninsula. Archives
of Biological Science 66(2): 743-763. https://doi.org/10.2298/ABS1402743R

Reed KM, Philips RB (1995) Molecular cytogenetic analysis of the double-CMA3 chromosome
in lake trout (Salvelinus namaycush). Cytogenetics and Cell Genetics 70(1—2): 104-107.
https://doi.org/10.1159/000134002

Sumner AT (1972) A simple technique for demonstrating centromeric heterochro-
matin. Experimental Cell Research 75: 304-306. https://doi.org/10.1016/0014-
4827(72)90558-7

Thiriot-Quiévreux C (2003) Advances in chromosomal studies of gastropod molluscs. Journal
of Molluscan Studies 69: 187-202. https://doi.org/10.1093/mollus/69.3.187

Thiriot-Quiévreux C, Insua A (1992) Nucleolar organiser region variation in the chromosomes
of three oyster species. Journal of Experimental Marine Biology and Ecology 157(1): 33-40.
https://doi.org/10.1016/0022-0981(92)90072-I

386 Jin-hai Wang @ Xiao-dong Zheng / Comparative Cytogenetics 12(3): 373-386 (2018)

Tiersch TR, Chandler RW, Wachtel SS, Elias S (1989) Reference standards for flow cytom-
etry and application in comparative studies of nuclear DNA content. Cytometry 10(6):
706-710. https://doi.org/10.1002/cyto.990100606

Vitturi R, Lafargue F (1992) Karyotype analyses reveal inter-individual polymorphism and as-
sociation of nucleolus-organizer-carrying chromosomes in Capros aper (Pisces: Zeiformes).
Marine Biology 112(1): 37-41. https://doi.org/10.1007/BF00349725

Wang HS, Luo X, You WW, Dong YW, Ke CH (2015) Cytogenetic analysis and chromosomal
characteristics of the polymorphic 18S rDNA of Haliotis discus hannai from Fujian, China.
PLoS One 10(2): e0113816. https://doi-org/10.1371/journal.pone.0113816

Wang JH, Zheng XD (2017) Comparison of the genetic relationship between nine Cepha-
lopod species based on cluster analysis of karyotype evolutionary distance. Comparative
Cytogenetics 11(3): 477-494. https://doi.org/10.3897/CompCytogen.v1 1i3.12752

Yoshida M, Ishikura Y, Moritaki T, Shoguchi E, Shimizu KK, Sese J, Ogura A (2011) Genome
structure analysis of mollusks revealed whole genome duplication and lineage specific re-
peat variation. Gene 483(1—2): 63-71. https://doi.org/10.1016/j.gene.2011.05.027

Zalesna A, Florek M, Rybacki M, Ogielska M (2017) Variability of NOR patterns in European
water frogs of different genome composition and ploidy level. Comparative Cytogenetics
11(2): 249-266. https://doi.org/10.3897/CompCytogen.v1 1i2.10804

Zhang XL, Onozato H (2004) Hydrostatic pressure treatment during the first mitosis does not
suppress the first cleavage but the second one. Aquaculture 240(1/4): 101-113. https://
doi.org/10.1016/j.aquaculture.2004.07.004